Proteins such as enzymes involved in physiological and pathological processes are important targets in the development of pharmaceutical compounds and treatments. Knowledge of the three dimensional (tertiary) structure of proteins allows the rational design of mimics or modulators of such proteins. By searching structural databases using structural parameters derived from the protein of interest, it is possible to select molecular structures that may mimic or interact with these parameters. It is then possible to synthesise the selected molecular structure and test its activity. Alternatively, the structural parameters derived from the protein of interest may be used to design and synthesise a mimic or modulator with the desired activity. Such mimics or modulators may be useful as therapeutic agents for treating certain diseases. For example, WO98/07835 discloses crystal structures of a protein tyrosine kinase optionally complexed with one or more compounds. The atomic coordinates of the enzyme structures and any of the bound compounds are used to determine the three-dimensional structures of kinases with unknown structure and to identify modulators of kinase functions. As another example, WO99/01476 discloses the crystal structures of anti-Factor IX Fab fragments (antibodies) and their use to identify and design new anticoagulant agents.
Knowledge of the three-dimensional structure of a protein is essential for the rational design of mimics or modulators of that protein. Lack of structural knowledge is a barrier to the development of new mimics or modulators that may have extremely useful pharmaceutical properties.
In Eukaryotes, the cell cycle is largely controlled by an ordered cascade of protein phosphorylation. Several families of protein kinases that play critical roles in this cascade have now been identified. The activity of many of these kinases is increased in human tumours when compared to normal tissue. This can occur by either increased levels of expression of the protein (as a result of gene amplification for example), or by changes in expression of co-activators or inhibitory proteins.
The first identified, and most widely studied of these cell cycle regulators have been the cyclin-dependent kinases (or CDKs). Activity of specific CDKs at specific times is essential for both initiation and coordinated progress through the cell cycle. For example, the CDK4 protein appears to control entry into the cell cycle (the G0-G1-S transition) by phosphorylating the retinoblastoma gene product pRb. This stimulates the release of the transcription factor E2F from pRb, which then acts to increase the transcription of genes necessary for entry into S phase. The catalytic activity of CDK4 is stimulated by binding to a partner protein, Cyclin D1. One of the first demonstrations of a direct link between cancer and the cell cycle was made with the observation that the Cyclin D1 gene was amplified and cyclin D1 protein levels increased (and hence the activity of CDK4 increased) in many human tumours (Reviewed in Sherr, 1996, Science 274: 1672–1677; Pines, 1995, Seminars in Cancer Biology 6: 63–72). Other studies have shown that negative regulators of CDK function are frequently down-regulated or deleted in human tumours, again leading to inappropriate activation of these kinases (Loda et al., 1997, Nature Medicine 3(2): 231–234; Gemma et al., 1996, International Journal of Cancer 68(5): 605–11; Elledge et al. 1996, Trends in Cell Biology 6; 388–392).
More recently, protein kinases that are structurally distinct from the CDK family have been identified which play critical roles in regulating the cell cycle and which also appear to be important in oncogenesis. These include the newly-identified human homologues of the Drosophila Aurora and S. cerevisiae Ipl1 proteins. Drosophila Aurora and S. cerevisiae Ipl1, which are highly homologous at the amino acid sequence level, encode serine/threonine protein kinases. Both Aurora and Ipl1 are known to be involved in controlling the transition from the G2 phase of the cell cycle through mitosis, centrosome function, formation of a mitotic spindle and proper chromosome separation/segregation into daughter cells. The three human homologues of these genes, termed Aurora A, B and C, encode cell cycle regulated protein kinases. These show a peak of expression and kinase activity at the G2/M boundary (Aurora A, C) and in mitosis and cytokinesis (Aurora B). Several observations implicate the involvement of human Aurora proteins, in particular Aurora A in cancer. The Aurora A gene maps to chromosome 20q13, a region that is frequently amplified in human tumours including both breast and colon tumours. Aurora A may be the major target gene of this amplicon, since Aurora A DNA is amplified and Aurora A mRNA over expressed in greater than 50% of primary human colorectal cancers. In these tumours Aurora A protein levels appear greatly elevated compared to adjacent normal tissue. In addition, transfection of rodent fibroblasts with human Aurora A leads to transformation, conferring the ability to grow in soft agar and form tumours in nude mice (Bischoff et al., 1998, The EMBO Journal. 117(11): 3052–3065). Other work has shown that artificial over expression of Aurora A leads to an increase in centrosome number and an increase in aneuploidy (Zhou et al., 1998, Nature Genetics. 20(2): 189–93).
Importantly, it has also been demonstrated that abrogation of Aurora A expression and function by antisense oligonucleotide treatment of human tumour cell lines (Bischoff and Ploughman, 1999, Trends in Cell Biology, 9(11): 454–459 or by a small molecule inhibitor of Aurora A kinase activity (Keen et al. 2001, poster #2455, American Association for Cancer Research annual meeting, New Orleans USA) leads to defects in mitosis, cell cycle arrest and exerts an antiproliferative effect in these tumour cell lines. This indicates that inhibition of the function of Aurora A will have an antiproliferative effect that may be useful in the treatment of human tumours and other hyperproliferative diseases.
In order to design inhibitors of Aurora A kinase, it is necessary to know the three-dimensional structure of Aurora A kinase, in complex with various lead compounds. To date, the three-dimensional structure of Aurora A kinase has not been available. Further, it has not been possible to obtain crystals of any part of Aurora of sufficient quality to allow determination of the structure of the kinase domain including the site of inhibition.